Our work over the last funding period has focused on characterization of P2Y receptor targeting in polarized cells. These studies established that seven of the eight known subtypes of P2Y receptors display polarized expression in epithelial cells, with four receptors at the basolateral membrane and three receptors at the apical membrane. We have identified and characterized the sorting signals in five of these receptors, which revealed novel mechanisms by which the receptors achieve polarized expression. These findings will be extended to define the physical and structural properties of the sorting signals in the C-terminal tails of P2Y1, P2Y4 , and P2Y14 receptors. The proteins that bind to P2Y receptor sorting signals and direct their polarized targeting are unknown. We will identify these proteins and define their roles in receptor targeting. Lipid microdomains are implicated in localization of proteins in endothelial and epithelial cells and in the regulation of signaling molecules, including P2Y receptors. We will elucidate the role(s) that lipid rafts and caveolae play in the localization and signaling responses of P2Y receptors and delimit the receptor domain(s) involved in these interactions. Successful completion of these projects will provide major new insights into the mechanistic/structural basis of cell targeting and regulation of signaling activities of a therapeutically important class of receptors in the context of a physiologically relevant cell model. The nucleotide-activated P2Y receptors are proven or potential therapeutic targets for treatment of lung diseases, hypertension, and stroke. In epithelial cells, these receptors are targeted to distinct membrane surfaces, where they regulate ion and water transport across the epithelial monolayer. Although cellular mistargeting of proteins is often associated with disease, the cellular mechanisms for proper protein localization are still not well understood. Our studies will provide novel and important information on the cellular mechanisms of P2Y receptor targeting and the regulation of P2Y receptor signaling activity in epithelial cells.